Role of the 70-kDa subunit of human replication protein a (I): Single-stranded dna binding activity, but not polymerase stimulatory activity, is required for dna replication

Dong Kyoo Kim, Evelyn Stigger, Suk Hee Lee

Research output: Contribution to journalArticlepeer-review

58 Scopus citations

Abstract

Replication protein A (RPA), also known as human single-stranded DNA-binding protein, is a three-subunit protein complex with multiple functions. Here, we investigated the role of the 70-kDa RPA subunit (p70) in DNA replication, by generating a series of deletion mutants. Mutant p70, which lacked 50 amino acids at the C-terminus, failed to interact with the 11-kDa RPA subunit (p11) and, when deleted further at the C terminus, was unable to interact with either the 34-kDa subunit (p34) or with p11, suggesting that p70 directly interacts with both p34 and p11. Studies with purified RPA mutants indicated that deletions at the N-terminal domain of p70 had very little effect on RPA's single-stranded DNA (ssDNA) binding activity, whereas deletion of amino acids 169-246 significantly weakened the DNA binding ability of RPA. By deleting amino acids 296-373 or 374-458, we totally abolished p70's ssDNA binding activity, suggesting that multiple p70 domains are involved in DNA binding. Two p70 domains, the N-terminal domain and the DNA binding domain, were required to stimulate DNA polymerase (pol) α, yet the DNA binding domain alone supported pol δ activity. Interestingly, RPA containing p70 with a zinc-finger domain deletion retained its DNA binding activity, but inhibited polα and δ activity. RPA that lacked ssDNA binding activity failed to support simian virus 40 (SV40) DNA replication in vitro, whereas mutant RPA that lacked pol α stimulatory activity (including the zinc-finger p70 mutant) functioned normally. We conclude that RPA's DNA binding activity, but not its pol α stimulatory activity, is required for DNA replication.

Original languageEnglish (US)
Pages (from-to)15124-15129
Number of pages6
JournalJournal of Biological Chemistry
Volume271
Issue number25
DOIs
StatePublished - 1996

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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